Emerging antibiotic resistance is making treatment of infections caused by Acinetobacter baumannii, Klebsiella pneumoniae and other Enterobacteriaceae increasingly ineffective. Many resistance genes are carried on mobile genetic elements and can readily be exchanged between strains and species. This project will use comprehensive genome analysis to improve our understanding of the genetic context of resistance genes, their chromosomal background, and lateral gene transfer. Properties of the drug resistance element transmission process in populations will be analyzed using historical and ongoing collections of isolates from several hospitals. Additional studies are designed to explore the role of mixed infections in genetic exchange and the relationship of pathogens to commensal bacteria in the microbiome. The scope and extent of DNA methylation in A. baumannii and K. pneumoniae will be analyzed using single molecule real time sequencing and the effect on gene expression will be determined. The results should be of value in development of molecular assays to facilitate diagnosis and selection of appropriate therapies for clinical infections.